Elongation Factor P (EF-P) is a universally conserved post-translationally modified protein that relieves ribosomal pausing at polyproline motifs by binding to the ribosome and entropically stimulating peptide bond formation. In all examples characterized to date EF-P and its homologs require post-translational modification to be functional. The function of EF-P modifications and the mechanism by which modifications would improve translation efficiency is unclear. Mutations in bacterial genes encoding EF-P (efp) or the corresponding modification pathways are highly pleiotropic, leading to a variety of detrimental phenotypes including slowed growth, loss of motility, attenuated virulence and hypersensitivity to antibiotics. Preliminary characterization of post-translational modification of Bacillus subtilis EF-P, which requires 5-aminopentanol addition for activity, challenges the notion that EF-P functions solely to maintain basic cellular function. In B. subtilis the ratio of modified to unmodified EF-P varies with growth phase, and neither deletion of EF-P nor removal of the modification impair vegetative growth, but instead specifically impair motility development. We hypothesize, based on the chemical diversity of permissive post-translational modification groups, that EF-P modification is regulatory. B. subtilis is an ideal model organism to explore this hypothesis as defects in either EF-P or EF-P modification impair swarming motility, a powerful phenotype for unbiased genetic selection. The objectives of this proposal are to uncover the structural and functional diversity of EF-P by investigating the different mechanisms by which this conserved, ubiquitous, translation factor functions during protein synthesis. Specifically, we will determine how EF-P acts as a cellular differentiation-specific translation factor and investigate the mechanism of translational control by EF-P.